Hematopoietic differentiation of human pluripotent embryonic stem cells

ABSTRACT

Disclosed herein are methods of obtaining human hematopoietic cells from human pluripotent embryonic stem cells using mammalian stromal cells. Hematopoietic cells derived in this way are useful for creating cell cultures suitable for transplantation, transfusion, and other purposes.

CROSS REFERENCES TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH BACKGROUND OF THEINVENTION

The present invention relates to the use of human embryonic stem cellsto create blood-related cells, and the use of those blood-related cellsfor various purposes.

Techniques for isolating stable cultures of human embryonic stem cellshave recently been described by our laboratory. See U.S. patent No.5,843,780 and J. Thomson et al., 282 Science 1145-1147 (1998). Thedisclosure of these publications and of all other publications referredto herein are incorporated by reference as if fully set forth below.

We have deposited two of our human embryonic stem cell lines with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209 U.S.A. on Jul. 7, 1999 and Jul. 15, 1999 respectively.Taxonomic descriptions of these deposits are human embryonic stem celllines H1 and H9 respectively. It has been proposed in these publicationsthat such cell lines may be used for, among other things, providing asource of specified cell lines of various types for research,transplantation and other purposes.

Under the storage and culturing conditions described in thesepublications the cell lines are maintained long term withoutdifferentiation into specific cell types. When the cell lines aresubsequently injected into immunodeficient mice, they form teratomasdemonstrating differentiation into multiple tissue types.

When ES cells are used to produce desired cells, it is often preferableto optimize differentiation towards specific cell types. In the case ofhematopoietic cells it is desirable that this result in hematopoieticcells that can be isolated and used to form multiple hematopoieticlineages. These cells may include, but not be limited to, hematopoieticstem cells.

Hematopoietic stem cell populations have been isolated directly frombone marrow. See C. Baum et al. 89 PNAS USA 2804-2808 (1992). However,this relies on a supply of bone marrow to obtain the cells.

There have also been some attempts to direct murine embryonic cellpopulations towards hematopoietic cells. See e.g. U.S. Pat. No.5,914,268; G. Keller, 7 Current Opinion In Cell Biology, 862-869 (1995);and T. Nakano et al. 265 Science 1098-1101 (1994). See also M. Weiss, 11Aplastic Anemia And Stem Cell Biology, 1185-1195 (1997); and S. Morrisonet al., 11 Annu. Rev. Cell Dev. Biol., 35-71 (1995).

However, applying these teachings to primates has proven difficult. Forexample, in F. Li et al., 92 Blood 368a (1998) there was a discussion oftechniques for differentiation of rhesus embryonic stem cell lines usinga stromal cell line and exogenous cytokines. However, that group hasmore recently reported that their techniques had inadequate formation ofcolonies.

The treatment of various diseases by tissue transplantation has becomeroutine. However, there can be waiting lists to obtain natural donatedorgans, cells, or tissue. Even when the natural donor material becomesavailable there is often a problem with rejection. Traditionalapproaches for suppressing an immune response of recipients havedrawbacks. For example, immunosuppressive drugs are costly and oftenhave side effects.

In WO 98/07841 there was discussed techniques of deriving embryonic stemcells that are MHC compatible with a selected donor (e.g. transplantinga donor nucleus into an enucleated oocyte, followed by derivation of thestem cells therefrom). The application suggested that the resultingcells could be used to obtain MHC compatible hematopoietic stem cellsfor use in medical treatments requiring bone marrow transplantation.

However, some diseases such as type 1 diabetes mellitus or multiplesclerosis involve an autoimmune response. For example, merelytransplanting pancreatic islets (which are MHC compatible to thediseased individual) to replace destroyed pancreatic islets will notprovide sufficient long term reduction in type 1 diabetes mellitus, asthe immune system of the host will still attack the transplanted islets.

It can therefore be seen that a need exists for techniques for causinghuman embryonic stem cell cultures to differentiate to desiredhematopoietic colonies. Further, it is desired to develop improved usesfor hematopoietic cells.

BRIEF SUMMARY OF THE INVENTION

In one aspect the present invention provides a method for obtaininghuman hematopoietic cells. One exposes a human embryonic stem cellculture to mammalian hematopoietic stromal cells so as to thereby createhuman hematopoietic cells. At least some of the human hematopoieticcells that are so created are CD34⁺ and/or are capable of forminghematopoietic cell colony forming units in methylcellulose culture.

CD34 is a standard marker for hematopoietic stem cells, as described inC. Baum et al. 89 PNAS USA 2804-2808 (1992) and S. Morrison et al., 11Annu. Rev. Cell Dev. Biol., 35-71 (1995). The property of capability offorming a colony forming unit is indicative that the cells have thedesired characteristics to form more differentiated hematopoieticlineages.

The stromal cells are preferably derived from bone marrow cells orembryonic yolk sac cells. Murine stromal cells may be used for thispurpose. However, primate stromal and other mammalian stromal cellsshould be suitable as well.

In another aspect the invention provides a human hematopoietic cellwhich was derived from a human embryonic stem cell culture in vitro, andis capable of forming hematopoietic cell colony forming units inmethylcellulose culture. As used in this patent, the term “derived” isintended to mean obtained directly or indirectly (e.g. through one ormore intermediates or passages).

In yet another aspect the invention provides a method of transplantinghuman cellular material into a human recipient host. One obtains humanhematopoietic cells which have been derived in vitro from an embryonicstem cell culture. One then obtains a selected human cellular materialother than hematopoietic cells, the selected non-hematopoietic materialhaving major histocompatibility complex compatibility to thehematopoietic cells. One then transplants both the hematopoietic cellsand selected human non-hematopoietic cellular material into the humanhost.

For example, one can obtain human hematopoietic cells which have beenderived in vitro from an embryonic stem cell culture (e.g. using thetechniques described below). One also obtains human pancreatic isletswhich have MHC compatibility to the hematopoietic cells. Both thehematopoietic cells and pancreatic islets are then transplanted into thehuman (preferably after the recipient's own bone marrow has beeninactivated).

The pancreatic islets can be obtained directly from a donor whose cellswere used to create the embryonic stem cell culture. Alternatively, asingle embryonic stem cell culture can be differentiated along twodifferent paths. In one process the above technique can be used tocreate hematopoietic stem cells. These cells should develop intomultiple hematopoietic lineages when transplanted into appropriatehosts. These lineages should include lymphocytes which would be tolerantof other cells derived from the same parental embryonic stem cells. Inanother process the stem cells would be directed towards pancreaticislets.

In another example one could supply oligodendrocytes to a human who hasa multiple sclerosis condition. One obtains human hematopoietic cellswhich have been derived in vitro from an embryonic stem cell culture(e.g. using a technique described below). One also obtains humanoligodendrocytes which have MHC compatibility to the bone marrow cellsand transplants both the bone marrow cells and oligodendrocytes into thehuman.

The same human whose genetic material was used to create the embryonicstem cell can be a donor for the oligodendrocytes. Alternatively, thesame embryonic stem cell culture can be differentiated along twoseparate paths to provide the two transplantable materials.

With respect to either disease (and potentially other autoimmunediseases) the immune and autoimmune rejection problems should be reducedby this technique. In this regard, the recipient's original bone marrowcan be totally or partially inactivated by radiation or chemical meansbefore the transplantation. Thereafter, it is replaced at least in partby the transplanted hematopoietic cells. The elimination/reduction ofthe original bone marrow reduces the body's ability to create anautoimmune response. The matching of the MHC of the replacement bonemarrow and the second transplantable material insures that the secondmaterial won't be rejected by the transplanted bone marrow.

Moreover, co-transplantation of hematopoietic cells and other tissue canbe done to promote acceptance of the second tissue (e.g. heart muscleplus hematopoietic cells for treating heart disease; hepatocytes plushematopoietic cells for treating liver disease). By creatinghematopoietic chimeras improved acceptance of tissues with similarlymatched MHC type can be obtained.

The present invention should be suitable to obtain a wide variety ofhematopoietic cells of interest, such as erythroid cells, granulocytecells, macrophages, lymphocyte precursors, monocytes, B cells, T cells,and the like. In this regard, colonies of differentiated ES cellsdevelop into hematopoietic colonies when harvested, separated intosingle cells, and plated into appropriate cultures. These coloniesdemonstrate the development of colony-forming cells which proliferateinto colony-forming units (including colony forming unit-erythroid(CFU-E), blast forming unit-eythroid (BFU-E), colony formingunit-macrophage (CFU-M), colony forming unit-granulocyte/macrophage(CFU-GM) and colony forming unithigh proliferative potential (CFU-HPP)).The identification of colony forming cells indicates the differentiationof embryonic stem cells into hematopoietic cells capable of expandinginto defined hematopoietic lineages under defined conditions.

The objects of the present invention therefore include providing:

(a) methods of the above kind for obtaining hematopoietic cells;

(b) cells derived using those methods; and

(c) methods for using those derived cells for transplantation,transfusion and other purposes. These and still other objects andadvantages of the present invention will be apparent from thedescription of the preferred embodiments that follows. However, theclaims should be looked to in order to judge the full scope of theinvention.

DETAILED DESCRIPTION Embryonic Stem Cell Culture

The previously described human ES cell line H1 was used for the majorityof experiments, albeit some of the following studies were done with thepreviously described ES cell lines H9 (or H9.2) with similar results.These cells were removed from frozen (liquid nitrogen) stocks of cellsderived from the original isolated and propagated cell line. The H1 EScells were grown in 6 well culture dishes (Nunclon, Fisher).

The dish was first coated with 0.1% gelatin solution (Sigma) for one ormore days in a 37° C./5% CO₂ incubator. After the one or more days, thegelatin solution was removed and the wells of the plate were next coatedwith irradiated mouse embryonic fibroblast (MEF) cells. MEF cells werederived from day 12-13 mouse embryos in medium consisting of DMEM(GibcoBRL) supplemented with 10% fetal bovine serum (Hyclone or Harlan),2 mM l-glutamine (GibcoBRL), and 100 units/ml. Penicillin, 100 mg/mlstreptomycin (Sigma).

The MEF cells were irradiated with 5500 cGy from a cesium source priorto plating in the wells. The MEFs were added at a density of 5×10⁴cells/ml, 2.5 ml/well. The plate coated with MEFs was then placed in 37°C./5% CO₂ incubator for one or more days until addition of ES cells.

ES cells were passed onto new MEFs at approximately 5-8 day intervals.The time depends on cell density and morphologic appearance ofdifferentiation. For passage, the medium in a well of ES cells wasremoved and 1-2 ml of medium containing 1 mg/ml collagenase IV in DMEM(GibcoBRL) was added. The plate was then placed at 37° C./5% CO₂ for5-20 minutes until the colonies of ES cells began to round up.

The well was then scraped with a 5 ml pipette to detach the ES cellsfrom the plate. The contents of the harvested well were placed in a 15ml conical tube (Fisher) and spun in a centrifuge at 1000 rpm for 5minutes. The medium was removed and 10 ml of fresh medium was added.This ES cell medium consists of F12/DMEM (GibcoBRL)) supplemented with20% serum replacement medium (GibcoBRL), 8 ng/ml of bFGF (GibcoBRL), 1%non-essential amino acid solution (GibcoBRL), 1 mM l-glutamine(GibcoBRL), and 0.1M β-mercaptoethanol.

The cells were again spun (5 min/1000 rpm), medium removed andresuspended at a concentration of 2.5 ml of medium for each (typically15 ml medium for plating into 6 new wells, this would be a 1:6 passage).The cells were then pipetted into the wells of a plate that had beenpreviously coated with MEFs as described above. The cells were evenlydistributed into each well and the plate was placed in an incubator at37° C./5% CO₂.

At times if there were colonies of ES cells showing morphologicappearance of differentiation prior to cell passage, these colonies wereremoved by gentle scraping with a pulled glass pipette. This was donewith observation through a dissecting microscope. After removal of thedifferentiated cells, the remaining colonies were passaged as above.

After passage, each well of ES cells was “fed” with fresh medium at24-48 hour intervals. Here, the medium of each well was removed and 2.5ml of fresh ES medium was added. All feeding and passage of ES cellswere done in a sterile environment.

Differentiation of ES Cells

To promote hematopoietic differentiation of the human ES cells, the EScells were harvested as above. The cells were then plated in 6 wellplates coated with a mammalian stromal cell. In one experiment we usedC166 cells that were previously irradiated with 2500 cGy. The C166 cellswere originally obtained from the yolk sac of mice at embryonic day 12and were graciously provided by Dr. Robert Auerbach (UW-Madison).

In another experiment, S17 cells were used. They were originallyobtained from mouse bone marrow, and were graciously provided by Dr.Kenneth Dorshkind (then at UC-Riverside, now at UCLA).

The C166 or S17 cells were plated at a density of 1×10⁵ cells/ml, 2.5ml/well. The ES cells plated onto either S17 of C166 cells were thenallowed to grow in a medium consisting of DMEM (GibcoBRL) supplementedwith 20% fetal bovine serum (Hyclone), 1% nonessential amino acidsolution, 0.1M β-mercaptoethanol, and 1 mM l-glutamine. This medium wasreplaced in each well at 24-72 hour intervals with fresh medium. Inselecting an appropriate medium, one merely needs to provideconventional conditions for cell growth, albeit supplemented with thespecified stromal cells.

After 3-7 days from plating onto S17 or C166 cells, the ES cells beganto visually appear differentiated in that they did not have the sameuniform appearance as the undifferentiated ES cells maintained on MEFfeeder cells. The colonies of ES cells began to form multiple differentcell types. Some of these colonies had regions that appeared to consistof cells with a cobblestone morphology indicative of colonies of earlyhematopoietic progenitor cells.

Confirming Blood-Related Cells

One method to determine the presence of appropriate hematopoietic cellsis to assay for hematopoietic colony forming cells (CFCs) in semisolidmethylcellulose-containing medium. Here, the ES cells were allowed todifferentiate on either C166 or S17 cells for 2-3 weeks, maintained asdescribed above. After this time the medium was removed. 2.5 ml ofcalcium and magnesium free phosphate buffered saline (PBS) was added for2-5 minutes, removed, and 1.5 ml. of trypsin (0.125%)-EDTA (1 mM) mediumwas added.

The cells were then placed at 37° C./5% CO₂ for 10 minutes. After thistime, the colonies began to disassociate. The cells were furtherdisassociated by pipetting and scraping the wells. The cells were placedin a 15 ml. conical, spun 5 min/1000 rpm, medium removed and 10 ml freshmedium (DMEM +10% FBS+l-glutamine+pen/strep) was added, and spun again.The cells were then suspended in 5 ml medium and passaged through a 100mM nytex filter to remove clumps of cells.

The filter was washed with an additional 5 ml medium. Thedisassociated/filtered cells were then counted on a hemacytometer and1×10⁶ (usually, but not always this many cells) cells were placed in anew 15 ml conical. These cells were then spun, medium removed and 5 mlmedium consisting of IMDM (GibcoBRL) supplemented with 2% fetal bovineserum (Hyclone) was added. Cells were spun, medium removed and 250 ulmedium (IMDM+2% FBS) was added.

In accordance with the specified test conditions, these cells were thenadded to 2.5 ml of Methocult GF+ H4435 medium (StemCell Technologies).This medium consists of 1.0% methylcellulose, supplemented with 30% FBS,20 ng/ml IL-3, 20 ng/ml IL-6, 50 ng/ml stem cell factor, 3 units/mlerythropoietin, 20 ng/ml GM-CSF, 20 ng/ml G-CSF, 2 mM 1-glutamine, 0.1mM b-mercaptoethanol, 1% bovine serum albumin. The cells inmethylcellulose were then vortexed vigorously and then 1.1 ml of themixture was plated onto a P35 plastic dish (Stem Cell Technologies),spread evenly on the dish and placed at 37° C./5% CO₂.

Duplicate plates of each sample were typically plated with 4×10⁵cells/plate. After 14-21 days, the plates were analyzed under amicroscope for the presence of hematopoletic colonies. The colonies wereidentified by comparison to a colony atlas (StemCell Technologies) orthe book: Culture of Hematopoietic Cells, RI Freshney, IB Pragnell, MGFreshney, eds., Wiley-Liss, Inc. 1994. Colonies were identified as oneof the following: colony forming unit-erythroid (CFU-E), blast formingunit-eythroid (BFU-E), colony forming unit-macrophage (CFU-M), colonyforming unit-granulocyte/macrophage (CFU-GM) or colony forming unit-highproliferative potential (CFU-HPP)

The presence of the desired hematopoietic cells can also be confirmed byflow cytometry. One can look for specified cell surface antigens by flowcytometry. Here, ES cells differentiated on S17 cells or C166 cells asdescribed above for 14-21 days, were harvested with trypsin/EDTA asdescribed above and passed through a 100 mM nytex filter. The filteredcells were counted on a hemacytometer, then aliquotted into 15×75plastic tubes (Fisher) at approximately 1×10⁵ cells/tube. The cells werethen spun, medium removed and 2-3 ml of FACS medium was added. (FACSmedium is PBS with 0.5% BSA (Sigma), 0.1% sodium azide (Sigma)).

The cells were again spun and medium removed. Next an antibody directlylinked to a fluorescent marker (FITC or PE) was added to the wells at aconcentration as recommended by the supplier. Cells have been analyzedwith the following antibodies: CD34-FITC (Immunotech), CD45-PE(Pharmingen). IgGl-FITC and IgGl-PE were used as isotype controls fornon-specific staining of the cells. Cells were incubated with theappropriate antibody for approximately 30 min on ice, washed 1-2 timeswith 2-3 ml FACS medium and resuspended in approximately 0.5 ml FACSmedium.

The antibody labeled cells were then analyzed using a FACScan (BectonDickinson) as per manufacturers recommendations. The presence of deadcells was determined by addition of propidium iodide (1 mg/ml solution,5 ul added per tube) or 7-AAD (Calbiochem) (0.2 mg/ml, 5 ul/tube). Thesoftware for analysis was either PC Lysis or Cellquest.

The following experimental techniques were used to analyze antigenexpression by immunohistochemistry (IHC). Here, differentiated ES cellsthat have been co-cultured with either C166 or S17 as above, wereharvested with trypsin/EDTA as above. The cells were resuspended inmedium containing DMEM supplemented with 10% FBS at a concentration ofapproximately 1×10⁴-×10⁵. “Cytospin” preparations of these cells werethen made by spinning 1×10³-1×10⁴ cells onto a glass slide(Superfrost/plus, Fisher) with a Cytospin II centrifuge (Shanndon).

These slides were then fixed with cold acetone and stored frozen at −20°C. For IHC staining the slides were thawed at room temperature and thecell pellet was outlined with a wax pen (DAKO). The cells were thenstained as follows using a Vectastain ABC kit (Vector Laboratories,Burlingame, Calif.), all incubations were at room temperature. 100-200ul PBS was added onto the cells for 5 minutes then removed. Vectastainblocking antibody solution (horse serum) was then added onto the cellsfor 15 minutes. The cells were then blotted dry and 100-200 ul ofprimary antibody solution was added. The primary antibodies were: IgG1(1 ug/sample, Sigma), anti-CD34 (0.5 ug/sample, Immunotech), anti-CD45(1 ug/sample, DAKO), anti-class I (1 ug/sample, gift from Dr. PaulLeibson, Mayo Clinic), anti-CD14 (1 ug/sample, Pharmingen), anti-CD31 (1ug/sample, Pharmingen).

Primary antibody was added for 30 minutes followed by PBS for 10minutes. Next, biotinylated anti-IgG antibody was added (Vectastain kit,solution B) for 30 minutes followed by PBS for 10 minutes. NextVectastain ABC solution was added for 30 minutes at room temperaturefollowed by PBS for 10 minutes. Next DAB solution (Vectastain) was addedfor 5 minutes followed by washing under running tap water for 10minutes. In some experiments, the slides were then counterstained withGill's hematoxylline solution (Vector labs) for 3 minutes followed bywashing with running tap water for 10 minutes. The slides were then airdried. Cells staining positive appear brown.

CD34⁺ was demonstrated within a mixed population of cells (about 1%)after 2-3 weeks. Even more importantly, differentiated ES cells wereshown to develop into hematopoietic colonies when harvested, separatedinto cells and plated into methylcellulose (semi-solid) cultures.

Transplantation

Currently hematopoietic cell transplantation is conducted clinicallyprimarily for patients who have received high dose chemotherapy fortreatment of malignancies. These patients typically receive aheterogeneous mixture of hematopoietic cells either from an autologousor allogeneic source. Human ES-derived hematopoietic stem cells will atminimum provide a more homogeneous cell population for hematopoieticcell transplantation.

Further, as discussed above, the MHC characteristics of thetransplantation can now be controlled, thereby enabling treatment ofautoimmune diseases. For example, both hematopoietic stem cells (HSCs)and a second lineage (e.g. pancreatic islets for diabetes oroligodendrocytes for multiple sclerosis) could be derived from the sameparental ES cell line. With both lineages available, a hematopoieticchimera could be first created by performing a fully allogeneichematopoietic cell transplant (HCT). The established state of chimerismwould allow the recipient's immune system to “see” the subsequenttransplant of the second cell type (e.g. pancreatic islets cell oroligodendrocyte) as “self” and should not be rejected.

Note for example that oligodendrocytes have been obtained from mouse EScells (0. Brustle et al., 285 Science 754-6 (1999)), as have cardiacmuscle cells (M. Klug et al., 98 J. Clin. Invest. 216-224 (1996)).

This method of creating hematopoietic chimeras will also promoteacceptance of tissues transplanted for reasons other than autoimmunity.In this regard, mice receiving allogeneic hematopoietic stem cells donot reject other tissues with the same genetic background as thehematopoietic cells, but will still reject third party grafts. See K.Gandy et al., 65 Transplantation 295-304 (1998).

In addition to animal studies, there are now clinical case reports ofhuman patients who have previously received a hematopoietic celltransplant later requiring a solid organ (kidney) transplant. In theseinstances, the kidney transplant from the same person who had previouslysupplied the bone marrow transplant is immunologically accepted withoutfurther immunosuppression. See T. Spitzer et al., 68 Transplantation480-484 (1999).

Work in canine models and more recently in human clinical trials hasshown that milder non-myeloablative conditioning regimens can be used tobetter prepare hosts for allogenic HCT. Here, only moderate doses oftotal body irradiation and a short course of immunosuppression are usedto prepare the hosts prior to receiving allogeneic HCTs.

Even though the preferred embodiments have been described above, it willbe appreciated by those skilled in the art that other modifications canbe made within the scope of the invention. For example, while twospecific stromal type cells have been selected for use, many others arealso suitable. For example, one publicly available stromal cell line isthe M2-10B4 cell line having ATCC designation number CRL-1972.

Further, while the above description focuses on the creation ofprecursors for red blood cells and bone marrow, various otherblood-related cells of interest can be obtained in quantity using theabove techniques. See also U.S. Pat. No. 5,914,268. Thus, the claimsshould be looked to in order to judge the full scope of the invention.

Industrial Applicability

The invention provides blood-related cells useful for transplantation,research and other purposes.

We claim:
 1. A method for obtaining human hematopoietic cells,comprising exposing a human pluripotent embryonic stem cell culture tomammalian hematopoietic stromal cells so as to thereby produce humanhematopoietic cells, wherein at least some of the human hematopoieticcells that are so produced will form hematopoietic cell colony formingunits if placed in methylcellulose culture.
 2. The method of claim 1,wherein at least some of the human hematopoietic cells that are soproduced are CD34⁺.
 3. The method of claim 1, wherein the stromal cellsare selected from the group consisting of bone marrow cells andembryonic yolk cells.
 4. A method for obtaining human hematopoieticcells, comprising exposing a human pluripotent embryonic stem cellculture in vitro to mammalian hematopoietic stromal cells so as tothereby produce human hematopoietic cells, wherein at least some of thehuman hematopoietic cells that are so produced will form erythroid blastforming units if placed in methylcellulose culture.
 5. The method ofclaim 4, wherein the human hematopoietic cells comprise at least somehematopoietic cells that are CD34⁺.
 6. A method of transplanting humancells into a human recipient host, comprising: obtaining humanhematopoietic cells which have been derived in vitro from a humanpluripotent embryonic stem cell culture; obtaining a selected human cellother than hematopoietic cells, the selected non-hematopoietic cellhaving major histocompatibility complex compatibility to the humanhematopoietic cells; and transplanting both the human hematopoieticcells and selected human non-hematopoietic cell into the human host. 7.The method of claim 6, wherein the selected human cell is a humanpancreatic islet.
 8. The method of claim 6, wherein the selected humancell is a human oligodendroeytcs.
 9. A method for obtaining humanhematopoietic cells, comprising exposing a human pluripotent stem cellculture to mammalian hematopoietic stromal cells so as to therebyproduce human hematopoietic cells, wherein at least some of the humanhematopoietic cells that are so produced will form hematopoietic cellcolony forming units if placed in methylcellulose culture.
 10. A methodof transplanting human cells into a human recipient host, comprising:obtaining human hematopoietic cells which have been derived in vitrofrom a human pluripotent stem cell culture; obtaining a selected humancell other than hematopoietic cells, the selected non-hematopoietic cellhaving major histocompatibility complex compatibility to the humanhematopoietic cells; and transplanting both the human hematopoieticcells and selected human non-hematopoietic cell into the human host.